The incidence of esophageal adenocarcinoma (EAC) has increased dramatically in the US and other Western countries over the past 40 years. EAC is an aggressive tumor that is typically diagnosed at late stage resulting in very poor overall survival. If detected early however, effective treatment options are available including ablation, endoscopic mucosal resection and esophagectomy, with or without chemotherapy and radiation. EAC typically arises in the setting of chronic gastroesophageal reflux disease with the associated development of esophageal columnar metaplasia known as Barrett's Esophagus (BE). BE is the strongest known risk factor for EAC and patients with BE are therefore recommended to enter endoscopic surveillance programs to detect dysplasia or early EAC. However, the use of repeat endoscopies with multiple biopsies is an expensive and invasive model for BE surveillance. We believe that combining esophageal cytology with next-generation sequencing to detect mutations that drive development of dysplasia and EAC will provide a novel, less invasive and more cost-effective approach to BE surveillance. In Specific Aim 1, we will test the feasibility of this hypothesis by combining an FDA approved device for esophageal cytology sample collection (the EsophaCap(tm)) with targeted, next-generation sequencing of a panel of ~100 genes known to be drivers of EAC development. This panel will be identified from whole exome sequence data on 250 EAC samples and RNA-seq data on 100 of the same tumors. This data is available to us through our previously published work (Dulak, et al. Nature Genetics 2013) and an ongoing study funded by Genome Canada. This data, along with access to the EsophaCap(tm) for immediate use in the USA puts us in a unique position to evaluate the feasibility of a molecular cytology test to detect disease progression in patients with BE. Specifically, in Aim 1A we will determine what proportion of EAC driver mutations, identified in multiple biopsy samples, are also detectable in matched cytology samples. This will determine overall feasibility for our proposed approach as the cytology samples will be contaminated with normal cells that will dilute the mutant allele fractions. In related aim 1B, we will also determin the mutation heterogeneity in BE by identifying mutations in multiple independent biopsies from the same patients. This will allow us to estimate the sequence depth required to detect all mutations in cytology samples and will also facilitate an analysis of clonal heterogeneity and evolution in BE. Specific Aim 2 will utilize a similar approach to address a critical question regarding ablation of BE; is the neosquamous epithelium that develops following ablation genetically normal or does it harbor mutations that could still incur cancer risk? This will be determined by comparing mutations in BE and neosquamous epithelium pre and post-ablation. This data will help determine the need for follow-up surveillance in BE patients treated with ablative therapy and will provide insight into the origin of stem cells that give rise to BE and neosquamous epithelium.